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Algal Pheromone Biosynthesis: Stereochemical Analysis and Mechanistic Implications in Gametes of Ectocarpus siliculosus Fabio Rui and Wilhelm Boland* Max Planck Institute for Chemical Ecology, Department of Bioorganic Chemistry Hans-Kn€ oll-Strasse 8, D-07745 Jena, Germany
[email protected] Received March 9, 2010
During sexual reproduction, female gametes or eggs of brown algae release pheromones to attract their male mating partners. The biologically active compounds comprise linear or alicyclic unsaturated hydrocarbons derived from the aliphatic terminus of C20 polyunsaturated fatty acids (PUFAs) by oxidative cleavage. The current study addresses the stereochemical course of the pheromone biosynthesis using female gametes of the marine brown alga E. siliculosus and chiral deuteriumlabeled arachidonic acids. The biosynthetic sequence is likely to proceed via an intermediary 9-hydroperoxyarachidonic acid, which is cleaved with loss of the C(16)-HR into the C11-hydrocarbon dictyopterene C and 9-oxonona-(5Z,7E)-dienoic acid.
Introduction 1
Brown algae live on marine coasts worldwide. They are distantly related to other eukaryotic groups such as plants and fungi, and they provide a valuable source of novel polysaccharides and lipids.2 Their lipid metabolites comprise not only complex and bioactive oxylipins3,4 but also C11 hydrocarbons deriving from the transformation of C20 polyunsaturated fatty acids (PUFAs).5 During sexual reproduction, (1) van den Hoek, C.; Mann, D. G.; Jahns, H. M.; van den Hoek, C.; Mann, D. G.; Jahns, H. M. Algae: An Introduction to Phycology; Cambridge University Press: Cambridge, 1995. (2) Charrier, B.; Coelho, S. M.; Le Bail, A.; Tonon, T.; Michel, G.; Potin, P.; Kloareg, B.; Boyen, C.; Peters, A. F.; Cock, J. M. New Phytol. 2008, 177, 319–332. (3) Gerwick, W. H.; Proteau, P. J.; Nagle, D. G.; Wise, M. L.; Jiang, Z. D.; Bernart, M. W.; Hamberg, M. Hydrobiologia 1993, 261, 653–665. (4) Ritter, A.; Goulitquer, S.; Salaun, J. P.; Tonon, T.; Correa, J. A.; Potin, P. New Phytol. 2008, 180, 809–821. (5) Stratmann, K.; Boland, W.; M€ uller, D. G. Angew. Chem., Int. Ed. Engl. 1992, 31, 1246–1248.
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mediated by eggs or sessile female gametes and flagellated male gametes, these compounds direct the motile male gametes to the signaling female. The relevant compounds are linear or alicyclic unsaturated C8-, C11-, or oxygenated C11-hydrocarbons6 (selected examples are shown in Figure 1). The structurally different C11 hydrocarbons 1-8 derive from the aliphatic terminus of polyunsaturated fatty acids (PUFAs) such as arachidonic acid (9) and eicosapentaenoic acid, both of which are abundant in the female gametes.7 The mechanistic details of this biosynthetic pathway have been elucidated with certain diatoms, as these unicellular organisms can be cultivated easily and produce the same compounds. Cell homogenates of the diatom Gomphonema parvulum synthesize hydrocarbons dictyopterene A (7) and hormosirene (2) from arachidonic and eicosapentaenoic acid.8 (6) Maier, I. Prog. Phycol. Res. 1995, 11, 51–102. (7) Schmid, C. E.; Muller, D. G.; Eichenberger, W. J. Plant Physiol. 1994, 143, 570–574. (8) Pohnert, G.; Boland, W. Tetrahedron 1996, 52, 10073–10082.
Published on Web 05/26/2010
DOI: 10.1021/jo1004372 r 2010 American Chemical Society
JOC Featured Article
Rui and Boland
SCHEME 1. Biosynthesis of C11 Hydrocarbons in the Diatom G. parvuluma
FIGURE 1. Structures of selected brown algal pheromones. a
As illustrated in Scheme 1, first a lipoxygenase activates the fatty acid to the (9S)-hydroperoxide 10 which in turn suffers an oxidative cleavage to the respective hydrocarbon and the polar fragment 9-oxonona-5(Z),7(E-)dienoic acid (11). In the diatom, the abstraction of the pro-R hydrogen from C(16) of 10 is the rate-determining step of the cleavage reaction, which generates an enantiomeric mixture of transdivinylcyclopropane (dictyopterene A) (7) along with a small amount of the linear hydrocarbon cystophorene (1). In the latter case, the oxidative cleavage involves the loss of a hydrogen atom from C(13).9 Mechanistic studies have also been conducted with the asteraceae Senecio isatidaeus, which produces C11 hydrocarbons by decarboxylation of a C12 fatty acid ultimately derived from linolenic acid.10 Previous studies with female gametes of E. siliculosus established the origin of the algal pheromones form arachidonic and eicosapentaenoic acid, but mechanistic details have not been addressed.11 The chemotactic system of the brown alga E. siliculosus is particularly interesting since the genuine pheromone pre-ectocarpene (6) is inactivated after a short time by a Cope rearrangement to ectocarpene (5).12 While the thermolabile pre-ectocarpene (6) (t1/2 = 21 min at 18 °C)12 attracts male gametes down to 5 pmol L-1, the rearranged ectocarpene (5) requires 2000-fold higher concentrations for attraction.13 To investigate the stereochemical and mechanistic aspects of algal pheromone biosynthesis, we administered chirally labeled deuterated arachidonic acids as metabolic probes to suspensions of female gametes of E. siliculosus and analyzed their transformation products by GLC-MS. The results (9) Hombeck, M.; Pohnert, G.; Boland, W. Chem. Commun. 1999, 243– 244. (10) Neumann, C.; Boland, W. Eur. J. Biochem. 1990, 191, 453–459. (11) Stratmann, K.; Boland, W.; Muller, D. G. Tetrahedron 1993, 49, 3755–3766. (12) Pohnert, G.; Boland, W. Tetrahedron 1997, 53, 13681–13694. (13) Boland, W.; Pohnert, G.; Maier, I. Angew. Chem., Int. Ed. Engl. 1995, 34, 1602–1604.
HPETE: hydroperoxyeicosatetraenoic acid.
allowed to reconstruct the biosynthetic sequence from the fatty acid substrate to the genuine pheromone. Results and Discussion In diatoms, the C11 hydrocarbons of the type dictyopterene A (7), pre-ectocarpene (6), as well as dictyotene (4), and ectocarpene (5) are produced from PUFAs by consecutive action of a lipoxygenase and a lyase. As illustrated in Scheme 1, the lipoxygenase generates a (9S)-hydroperoxide that is cleaved by a lyase activity into a hydrocarbon and 9-oxonona-5Z,7E-dienoic acid (11). In diatoms, the mechanistic details were obtained by administration of labeled arachidonic acid to growing cultures of G. parvulum. In the present study, we followed the previous strategy and first developed a novel route to chirally labeled (16R)- and (16S)-[16,19,20-2H3]-arachidonic acids. Two additional deuterium atoms at C(19) and at C(20) of the probe were introduced to warrant a safe detection of the metabolites even after removal of the deuterium atom at C(16). As illustrated in Scheme 2, the scaffold of the chiral, deuterated arachidonic acid (16R)-[2H3]-9 was envisaged to be constructed by double-Wittig olefination with the bis-ylide of (Z)-hex-3-enyl-1,6-bis(triphenylphosphonium bromide) 12 and two easily accessible aldehydes.14,15 This strategy allows the simultaneous generation of a segment with three methyleneinterrupted Z-double bonds which is typical for highly unsaturated fatty acids. The deuterium-labeled hexanal 13 was synthesized as outlined in Scheme 3. Jacobsen’s hydrolytic kinetic resolution of benzylglycidyl ether 1416 provided the chiral building block (2S)-14 at a gram scale. Regioselective (14) Pohnert, G.; Boland, W. Eur. J. Org. Chem. 2000, 2000, 1821–1826. (15) Pohnert, G.; Adolph, S.; Wichard, T. Chem. Phys. Lipids 2004, 131, 159–166. (16) Schaus, S. E.; Brandes, B. D.; Larrow, J. F.; Tokunaga, M.; Hansen, K. B.; Gould, A. E.; Furrow, M. E.; Jacobsen, E. N. J. Am. Chem. Soc. 2002, 124, 1307–1315.
J. Org. Chem. Vol. 75, No. 12, 2010
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JOC Featured Article SCHEME 2. Synthesis of (16R)-[16,19,20-2H3]-Arachidonic Acid (16R)-[2H3]-9a
Rui and Boland SCHEME 3.
Synthesis of Chiral, Deuterium-Labeled Hexanal 13a
a Reagents and conditions: (a) (1) KN[Si(CH3)3]2, THF, -78 °C, (2) oxoester 19, (3) aldehyde 13; (b) LiOH, THF, H2O.
opening of the epoxide at C(3) with allylmagnesium bromide in the presence of CuI at low temperatures17 provided (2S)1-(benzyloxy)hex-5-en-2-ol 15. The enantiomeric purity of 15 (er=96.3 ( 0.1) was determined by GLC-MS of diastereoisomeric carbamates formed by 15 with 1-phenylethyl isocyanate.18 Stereospecific introduction of the deuterium label was achieved by a two-step mesylation/reduction procedure using LiAl2H4 without isolating the reactive mesylate.19,20 The displacement of the mesylate of a secondary alcohol by a deuteride from LiAl2H4 proceeds with complete inversion of configuration at the stereocenter.20 To confirm inversion at the stereocenter also in the mesylate of the benzyloxy alcohol 15, the absolute configuration of deuterated hexanol 18 was determined by 1H NMR after oxidation21 to deuterated hexanoic acid and esterification with methyl (S)-mandelate.22 If a chiral R-deuterated carboxylic acid is esterified with (S)-mandelate, the acid pro-S proton resonates at high fields of the pro-R proton.22 1H NMR of the methyl (S)-mandelate diester of [2,5,6-2H3]-hexanoic acid displays a major signal at 2.20-2.10 ppm and a small signal at 2.30-2.30 ppm, confirming the (R)-configuration of 18. The optical purity of 18 cannot be determined with this method because the procedure for the oxidation to hexanoic acid promotes enolization of the labeled aldehyde intermediate. The additional two deuterium atoms were introduced in the chiral, deuterated benzyl ether 16 by deuteration with Wilkinson’s catalyst23 to avoid deuterium scrambling.24 The benzyl group of 1-((2R)-[2,5,6-2H3]-hexyloxy)methylbenzene (17) was removed by hydrogenolysis with Pd(C) yielding (17) Huynh, C.; Derguini-Boumechal, F.; Linstrumelle, G. Tetrahedron Lett. 1979, 20, 1503–1506. (18) Habel, A.; Spiteller, D.; Boland, W. J. Chromatogr. A 2007, 1165, 182–190. (19) Abad, J. L.; Serra, M.; Camps, F.; Fabrias, G. J. Org. Chem. 2007, 72, 760–764. (20) Abad, J. L.; Camps, F.; Fabrias, G. J. Org. Chem. 2000, 65, 8582– 8588. (21) Thottumkara, A. P.; Bowsher, M. S.; Vinod, T. K. Org. Lett. 2005, 7, 2933–2936. (22) Parker, D. J. Chem. Soc., Perkin Trans. 2 1983, 83–88. (23) Osborn, J. A.; Jardine, F. H.; Young, J. F.; Wilkinson, G. J. Chem. Soc. Inorg. Phys. Theor. 1966, 1711–1732. (24) Thyman, J. H. P.; Crombie, L. Synthesis in Lipid Chemistry; The Royal Society of Chemistry: Cambridge, 1996.
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a
Reagents and conditions: (a) Jacobsen’s (R,R) catalyst, H2O; (b) CH2dCHCH2MgBr, CuI, THF, -40 °C; (c) Et3N, MsCl, CH2Cl2, -78 °C; (d) LiAl2H4, THF, -78 °C; (e) Wilkinson’s catalyst,2H2, benzene; (f) Pd-C, H2, CH2Cl2; (g) IBX, CH2Cl2, reflux.
(2R)-[2,5,6-2H3]-hexanol (18) without concomitant scrambling. Oxidation with IBX25 in CH2Cl2 afforded the labeled hexanal 13 and allowed to isolate the volatile aldehyde in moderate yield. The second carbonyl component, namely methyl 8-oxooct-(5Z)-enoate (19), was obtained from commercially available δ-valerolactone 20 by a one-pot methanolysis and oxidation (Scheme 4, see the Supporting Information). Methyl 5-oxopentanoate 21 was olefinated with the C3-homologating agent 2-(1,3-dioxan-2-yl)ethyltriphenylphosphonium bromide (22),26 resulting in methyl 7-(1,3-dioxan2-yl)hept-(5Z)-enoate (23). Methanolysis gave the corresponding dimethyl acetal 24, and hydrolysis in pentane/formic acid resulted in the target β,γ-unsaturated 8-oxoester 19 with the correct stereochemistry (Z/E ratio >95:5, GLC-MS). The sequential addition14 of carbonyl 19 and 13 to a solution of the bis-ylide of 12 gave the labeled fatty acid methyl ester 25 after HPLC purification. The low yield of the double olefination could be possibly due to the instability of the homoconjugated ester ylide intermediate formed by the bis-ylide of 12 and β,γunsaturated oxoester 19, as preliminary experiments with non labeled, saturated carbonyls gave higher yields. Methyl ester 25 was hydrolyzed in small batches shortly before the incubation experiments in order to avoid autoxidation, and resulted in (16R)-[16,19,20-2H3]-arachidonic acid (16R)-[ 2H3]-9. The metabolic probe (16R)-[2H3]-9 was obtained with good isotopic and stereoisomeric purity. The isotopic purity (d2:d3:d4 = 1:98:1) of the fatty acid methyl ester 25 was determined by GLC-MS comparing the molecular ion traces of the d1-d5 isotopomers corrected for the contribution of the natural abundance of 13C. Transformation of (16R)- and (16S)-[16,19,20- 2H 3]Arachidonic Acids in Gametes of Ectocarpus siliculosus. The typical volatiles released from suspensions of female gametes of E. siliculosus were ectocarpene (>95%), dictyotene (25) More, J. D.; Finney, N. S. Org. Lett. 2002, 4, 3001–3003. (26) Stowell, J. C.; Keith, D. R. Synthesis 1979, 132–134.
JOC Featured Article
Rui and Boland SCHEME 4.
Reaction Pathways and Kinetic Isotope Effect (KIE) in E. siliculosus
SCHEME 5. Biosynthesis of Dictyotene (4) in Gametes of E. siliculosus
FIGURE 2. GLC separation of (6R)- and (6S)-dictyotene. GLCseparation of dictyotene enantiomers was achieved on Hydrodex-β6-TBDMS: (a) dictyotene from gametes of E. siliculosus; (b) dictyotene reference from G. parvulum.
(ca. 3-4%), and trace amounts of the linear hydrocarbon cystophorene (
